Coxiella burnetii is an extremely infectious, intracellular bacterium that causes Q fever in humans and is classified as a select agent. Q fever typically presents as a debilitating, flu-like illness accompanied by pneumonia or hepatitis, but in a minority of cases a severe, chronic infection occurs with life-threatening endocarditis as the predominant manifestation. Little is known about Coxiella's virulence determinants or how the bacterium regulates events associated with its biphasic life cycle and infection of host cells, despite the central role that these activities play in survival. Our long-term goal is to elucidate the RNA-mediated regulation of these processes. Preliminary studies by our group have begun to analyze small RNAs (sRNAs) of large-cell variants (LCVs; stationary phase) and small-cell variants (SCVs; exponential phase) of the pathogen, and we have identified 6S RNA as a prominent sRNA that it is expressed at levels that are ~7-fold higher in SCVs relative to LCVs. We hypothesize that sRNAs play key roles in regulating Coxiella's growth, development and infection of host cells. These hypotheses will be addressed in two specific aims. In aim 1, we will identify sRNAs involved in regulating Coxiella's growth and development by characterizing sRNA profiles of SCVs and LCVs using high-throughput (deep) sequencing technology. Second, we will analyze infection-specific sRNAs of Coxiella by identifying and comparing sRNAs of bacteria grown in the context of host cells to those obtained from axenic (host cell-free) cultures. Results of aim 1 will provide a foundation to formulate Coxiella's sRNA networks and elucidate how they regulate development and intracellular parasitism. In aim 2, we will investigate the regulatory role of 6S sRNA in Coxiella's growth and development. 6S RNA is not a major repressor of stationary-phase genes in closely-related Legionella pneumophila, rather, the 6S RNA positively regulates several genes that enhance intracellular growth and virulence. This observation, together with a report showing that ss is a dominant regulator during exponential growth of Coxiella, leads us to hypothesize that 6S sRNA regulates both DNA-dependent RNA polymerase associated with sigma-70 (RNAP-s70) and RNAP-ss, with a preference for the latter complex. To address this hypothesis, we will elucidate Coxiella's 6S RNA interactions with RNAP, the individual s factors, RNAP-s70 and RNAP-ss at the molecular level, by biochemical and gel retardation studies. Second, we will conduct RNA-seq transcriptome analyses to identify Coxiella genes that are differentially expressed in response to excess or reduced levels of 6S RNA. The 6S RNA levels will be manipulated by stable shuttle vectors that transcribe 6S sense or antisense RNAs. Expression of 6S (anti)sense RNA will be repressed by a cis-lacIQ gene on the vector and induced with IPTG. RNA-seq results will allow us to formulate a 6S RNA regulatory network and improve the current state of knowledge regarding global transcriptional regulation in Coxiella. Moreover, the results will clarify the rol of 6S sRNA in g-proteobacteria, where orthologous s factors play distinctive roles in different bacterial species.